A human liver microphysiology platform and self assembly liver acinus model and methods of their use

ABSTRACT

Microfluidic devices for modeling three-dimensional tissue structures and methods for making and using the same are described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage filing under 35 U.S.C. §371 ofInternational application Ser. No. PCT/US2015/053361 entitled “A HumanLiver Microphysiology Platform And Self Assembly Liver Acinus Model AndMethods Of Their Use,” and filed on Sep. 30, 2015, which claims benefitof and priority under 35 U.S.C. §119(e) to U.S. Provisional ApplicationNo. 62/057,625 entitled “A Human Liver Microphysiology Platform And SelfAssembly Liver Acinus Model And Methods Of Their Use,” filed Sep. 30,2014. The contents of each of these applications are incorporated hereinby reference in their entireties.

STATEMENT OF GOVERNMENT INTEREST

The United States Government may have certain rights to this inventionpursuant to Grant No. 1UH2TR00503-01 entitled “A 3D Biomimetic LiverSinusoid Construct for Predicting Physiology and Toxicity” awarded bythe National Institutes of Health.

BACKGROUND

In drug discovery, as well as industrial and environmental health andsafety, there is a significant need for lower cost, easy-to-use, robust,high-throughput biological models that predict human safety andefficacy. Four major issues have limited the success of liver models aspredictors of pharmacokinetics (PK), toxicity and efficacy in humans: 1)lab animal models correlate poorly (˜50%) with human liver drug effects;2) human liver tissue is difficult to obtain and usually of variablequality; 3) simple 2D models do not replicate key functions associatedwith liver failure, such as fibrosis and the immune response; and 4) invitro models have exhibited a short functional lifetime (˜2 weeks) thatexcludes some chronic effects.

Recently established surgical research networks and cryopreservationtechniques have improved the quality of available human cells. Inaddition, significant progress has been made in developing inducedpluripotent stem cells (hereafter, “iPSC's”) into adult hepatocytes. Theability to put patient-specific cells into this in vitro model willdramatically expand its power to investigate liver diseases such asnonalcoholic fatty liver disease, infectious diseases, liver cancer anda niche site for metastatic breast cancer, as well as use in targeteddrug discovery and safety testing. Combined with recent developments inmicrofluidics, we are now able to construct an all human-cell,self-assembly, 3D, microfluidic model of the liver that can bemaintained for at least a month, referred to here as the self-assemblyliver acinus (SALA) model. In addition, a whole human livermicrophysiology platform (HLMP) has been developed that includes theSALA model in a microfluidic device, fluorescence-based biosensors forreal-time measurements, and a database for management, processing, andcomputational modeling of large and complex data sets. The SALA modeland platform (HLMP) disclosed here are novel in the use of multiplehuman cell types that self-assemble into an organ model that maintainsmany organizational and functional features of the liver in amicrofluidic device that allows imaging analysis of cellular responsesusing genetically engineered biosensors.

The media flow through the model provides physiological flow properties,and a means for delivering both nutrients and test compounds. The effluxmedia provides an opportunity to measure standard clinical biomarkersand mass spectrometry analysis of compound metabolism. Themultiparameter data generated by the model is accumulated in a database,where it is correlated with published data from preclinical, clinicaland post-market drug trials, in order to construct classifiers that willpredict human organ interactions. The SALA and HLMP comprise the cells,the microfluidic device, the media and other reagents, the biosensors,the image analysis algorithms, the Microphysiology database, and thepredictive models. This model and platform have the potential torevolutionize safety testing by providing a 3D microphysiology liverthat responds to compound exposure with many of the same mechanisms as ahuman liver, while allowing direct imaging of those mechanisms in“sentinel” cells with integrated biosensors, along with measurement ofstandard clinical biomarkers, to generate data in vitro that ispredictive of in vivo pharmacokinetics, pharmacodynamics (PK/PD), andefficacy testing in disease models.

SUMMARY

Embodiments of the invention are directed to a microfluidic deviceincluding a housing having at least one inlet and at least one outlet,extracellular matrix proteins disposed on at least one surface of thehousing, cells of at least two types associated with the extracellularmatrix proteins forming three-dimensional structures within the housing;and a flow medium contacting the cells. In some embodiments, the cellsof at least two types may be primary liver cells, cultured liver cells,induced pluripotent stem cells, primary hepatocytes, endothelial cells,immune cells, stellate cells, and combinations thereof. In certainembodiments, the cells of at least to types may be human hepatocytes,human endothelial cells, human immune cells, and human stellate cells.In particular embodiments, the cells may further include geneticallymodified cells expressing a genetically encoded fluorescence basedbiosensors, and the genetically encoded fluorescence based biosensor maydependent upon, for example, calcium levels, pH, glutathione levels,mitochondrial calcium levels, oxidative stress, or reactive oxygenspecies. In particular embodiments, expression of the geneticallyencoded fluorescence based biosensor may be in response to apoptosis,change in mitochondrial membrane potential, cell proliferation, freecalcium ion concentration, cell motility, and oxidative stress response.In certain embodiments, the three-dimensional structures may exhibitzonation, and in some embodiments, the three-dimensional structures maybe liver acini.

In some embodiments, the microfluidic device may further include a pumpfluidly connected to the inlet configured to propel flow medium throughthe housing. In various embodiments, the housing may include one or morechambers and one or more passages fluidly connecting the chambers, andin some embodiments, the extracellular matrix proteins may be disposedin the chambers of the housing. In particular embodiments, themicrofluidic device may further include a sensor positioned to detectanalytes in effluent exiting the housing, and in some embodiments, themicrofluidic device may further include an imager positioned to imagethe three-dimensional structures. In further embodiments, themicrofluidic device may include one or more ports positioned tointroduce substances into the flow medium.

Other embodiments are directed to a method for self-assembly of liveracini including the steps of providing a surface coated with anextracellular matrix protein; contacting the surface with hepatocytes;incubating the surface for about 12 to about 16 hours; contacting thesurface with endothelial cells, immune cells, or combinations thereof;incubating the surface for about 1 to about 12 hours; and contacting thesurface with polymerized collagen. The surface may be disposed within amicrofluidic device. In some embodiments, contacting the surface withpolymerized collagen may include introducing collagen into themicrofluidic device; inverting the microfluidic device; polymerizing thecollagen; and inverting the microfluidic device. In various embodiments,the hepatocytes may include genetically modified hepatocytes expressinga genetically encoded fluorescence based biosensors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a microfluidic device.

FIG. 2 is a diagram showing self-assembly of three-dimensional livertissue in the SALA model.

FIG. 3 are fluorescence micrographs of a microfluidic device includingself-assembled three-dimensional liver tissue in the SALA model.

FIG. 4 are data plots showing the long-term health and stability ofhepatocytes in the SALA model.

FIG. 5 are liquid chromatography-mass spectrometry chromatograms ofmetabolic clearance and metabolite production in the SALA model.

FIG. 6 are data plots form live cell monitoring of hepatocellularcytochrome C release biosensor and biochemical assays performed on theefflux media showing time and dose dependent toxicity an assessment ofacute nimesulide toxicity in the SALA model.

FIG. 7 are data plots showing an assessment of apoptosis resulting fromnimesulide toxicity in the SALA model.

FIG. 8 are data plots showing active immune function in the SALA model.

FIG. 9 are data plots showing activation and migration of stellate cellsin the SALA model.

FIG. 10 is a data plot showing bile efflux in vehicle and troglitazonetreated SALA model and micrographs showing bile efflux.

FIG. 11 is data plot showing the correlation of propidium iodide uptakewith cell area of hepatocytes in the SALA model.

FIG. 12 are micrographs showing stellate cell activation in the SALAmodel.

FIG. 13 are micrographs and a data plot showing uniform flow of flowmedium through the microfluidic device. Panel A shows fluorescence washout, and panel B shows a plot of dextran intensity in the effluent.

FIG. 14 are micrographs and a data plot showing the maintenance of twosubpopulations of cells in the SALA model using a microfluidic device.

FIG. 15 are micrographs and a data plot showing nefazodone inducedoxidative stress response in the SALA model using a microfluidic device.

DETAILED DESCRIPTION

With respect to the use of substantially any plural and/or singularterms in this disclosure, those having skill in the art can translatefrom the plural to the singular and/or from the singular to the pluralas is appropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth in thisdisclosure for sake of clarity.

It will be understood by those within the art that if a specific numberof an introduced claim recitation is intended, such an intent will beexplicitly recited in the claim, and in the absence of such recitationno such intent is present. For example, as an aid to understanding, thefollowing appended claims may contain usage of the introductory phrases“at least one” and “one or more” to introduce claim recitations.However, the use of such phrases should not be construed to imply thatthe introduction of a claim recitation by the indefinite articles “a” or“an” limits any particular claim containing such introduced claimrecitation to embodiments containing only one such recitation, even whenthe same claim includes the introductory phrases “one or more” or “atleast one” and indefinite articles such as “a” or “an” (for example, “a”and/or “an” should be interpreted to mean “at least one” or “one ormore”); the same holds true for the use of definite articles used tointroduce claim recitations. In addition, even if a specific number ofan introduced claim recitation is explicitly recited, those skilled inthe art will recognize that such recitation should be interpreted tomean at least the recited number (for example, the bare recitation of“two recitations,” without other modifiers, means at least tworecitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (forexample, “ a system having at least one of A, B, and C” would includebut not be limited to systems that have A alone, B alone, C alone, A andB together, A and C together, B and C together, and/or A, B, and Ctogether, etc.). It will be further understood by those within the artthat virtually any disjunctive word and/or phrase presenting two or morealternative terms, whether in the description, claims, or drawings,should be understood to contemplate the possibilities of including oneof the terms, either of the terms, or both terms. For example, thephrase “A or B” will be understood to include the possibilities of “A”or “B” or “A and B.”

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed in this disclosure also encompass any and all possiblesubranges and combinations of subranges thereof. Any listed range can beeasily recognized as sufficiently describing and enabling the same rangebeing broken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed in thisdisclosure can be readily broken down into a lower third, middle thirdand upper third, etc. As will also be understood by one skilled in theart all language such as “up to,” “at least,” and the like include thenumber recited and refer to ranges which can be subsequently broken downinto subranges as discussed above. Finally, as will be understood by oneskilled in the art, a range includes each individual member.

Embodiments of the invention are directed to microfluidic deviceincluding a self-assembled liver acinus, and methods for using suchmicrofluidic devices for in vivo pharmacokinetics, pharmacodynamics(PK/PD), and efficacy testing in disease models. In various embodiments,the microfluidic device may include a housing and multiple human celltypes and a flow of medium disposed within the housing. In someembodiments, the microfluidic device may include other components thatreflect in vivo biology of the liver or other organs disposed within thehousing. The microfluidic device may further include a variety ofsensors positioned to detect discharged biomaterials such as metabolitesand other compositions produced by the cells in the microfluidic deviceover time. The methods of various embodiments may generally include thesteps of introducing a compound or composition under study into themicrofluidic device and detecting discharged biomaterials. In someembodiments, the device may include a computer or other means foranalyzing the detected discharged biomaterials. Thus, embodiments of themethod include identifying the discharged biomaterials. The microfluidicdevice may function for one month or more to measure chronic effects ontoxicity and disease progression.

A diagram showing an example of the microfluidic devices of variousembodiments is provided in FIG. 1. The microfluidic device 10 of FIG. 1includes a housing 100 having various fluidly connected chambers 102 andpassages 104. The housing 100 of various may be clear for imaging thecells and tissues in the microfluidic device, or at least a portion ofthe housing may be clear. In some embodiments, the housing 100 mayinclude upper and lower substrates and either the upper or lower or boththe upper and lower substrates may be optically transparent, i.e.,clear. In other embodiments, the housing may include transparent windowsare various locations allowing for observation of the cells and tissuesenclosed within the housing at those locations. The housing may includeone or more passages 104 for cellular adhesion and tissue growth, and insome embodiments, the housing may include one or more chambers 102connected by passages 104. The chambers 102 may be configured to supporttissue growth. For example, the chambers 102 may be coated with collagenor other extracellular matrix components that facilitate adherence ofcells of various tissue types to surfaces of the chambers. Passages 104may not include such coatings to discourage cell adherence and growth tomaintain flow of flow medium. The passages 104 and chambers 102 ofvarious embodiments may be configured to provide a continuous flow offlow medium from an inlet 106 to an outlet 108 of the device.

In some embodiments, the microfluidic device may be self-contained (notdepicted) meaning that the cells, extracellular matrix proteins, andflow medium are loaded into the microfluidic device, and themicrofluidic device is sealed creating a closed system. In otherembodiments, the microfluidic device 10 may have at least one inlet 106and at least one outlet 108 through which flow medium may be added to orremoved from the microfluidic device, which contains established livinghuman cells. In certain embodiments, the microfluidic device 10 maymimic directional blood flow and provide an in vivo-like route of drugadministration, nutrient delivery, and oxygen replenishment, necessaryfor establishing metabolic zonation and an elimination route formetabolic waste and other secretions. Such a flow may provide an effluxmedia for standard clinical measurements of organ function, for example,albumin and urea synthesis, and evidence of drug challenge such asenzyme secretion, drug clearance, and metabolites.

In some embodiments, the microfluidic device 10 may include one or moresensors 110 configured to monitor discharged biomaterials created frommaterials introduced into the microfluidic device. For example, in someembodiments, a drug or potentially therapeutic compound can beintroduced into the microfluidic device, and the one or more sensors 110may be configured to detect drug metabolites and other biochemicalby-products produced as a result of the introduction of the drug intothe device in an efflux media. In such embodiments, the microfluidicdevice 10 may provide a means for testing the effect of the drug onspecific tissue types such as liver acini. Embodiments are not limitedto particular sensors 110, and the microfluidic device may includesensors such as, biochemical, mass spectroscopy, fluorescence-basedbiosensors, and the like and combinations thereof. Such sensors maygenerally be non-destructive meaning they do not physically touch thecells or extracellular matrix proteins in the microfluidic device. Incertain embodiments, such non-destructive sensors may include opticalsensors such as those typically used for secretion assays. In particularembodiments, the sensors may be real-time meaning that the deviceidentifies and/or measures the discharged biomaterials as they aredischarged into the flow medium, and in some embodiments, the sensorsmay provide quantitative high content analysis (HCA) optical read-outs.In certain embodiments, the microfluidic device 10 may monitor any ofthe various cell types disposed within the microfluidic deviceindividually or in user-defined combinations.

In some embodiments, the microfluidic device 10 may include one or moreimaging devices 112 positioned to capture images of the cells andtissues disposed within the housing 100. The imaging device 112 may bein a fixed position, and in other embodiments, the imaging device 112may be movable.

The microfluidic device 10 may include any number of cells of varioustypes, which may be chosen to mimic particular tissue-types, and in someembodiments, the microfluidic device may include extracellular matrixproteins or other components of the organ of interest that allow thecells to organize into the particular tissue-type. For example, in someembodiment, the microfluidic device may include liver cells and amixture of extracellular matrix proteins disposed within the housing ofthe microfluidic device. In some embodiments, the extracellular matrixproteins may self-assembly into three-dimensional scaffolding necessaryto produce the tissue, such as liver acini. In other embodiments, thehousing may include a projections or other surfaces onto which theextracellular matrix proteins can adhere to produce thethree-dimensional architecture necessary to mimic the tissue type ofinterest. In certain embodiments, the microfluidic device may beconfigured to mimic liver tissue, and the extracellular matrix proteinsmay be selected from extracellular matrix proteins necessary to produceliver acini. One or more types of liver cells may be adhered to theextracellular matrix proteins to form the acini, and in particularembodiments, at least four human liver cell types may be containedwithin the microfluidic device to mimic acini. In certain embodiments,the human liver cells may be derived from a patient.

In example embodiments, the cells contained within the microfluidicdevice may be replatable cryopreserved human hepatocytes. In otherexample embodiments, the cells contained within the microfluidic devicemay be hepatocytes and three non-parenchymal cell (NPC) establishedhuman cell culture lines for the endothelial, immune, and stellatecells. These cell types can be chosen for expression of particularphenotypes. For example, in some embodiments, the microfluidic devicemay include LX-2 stellate cells that express human collagen necessaryfor the fibrotic scarring and U937 monocytic cells that expressesreceptors necessary for immune mediated toxicity. In certain embodimentsthe cells contained within the microfluidic device may be cells derivedfrom patients. Various patients having different genetic backgrounds maybe used to source the cells. For example, the microfluidic devices ofvarious embodiments may contain cells from patients having normal liverfunction or patients having non-alcoholic fatty liver disease, cancer,or other liver specific diseases. Such microfluidic devices may provide,as well as toxic response to drugs different will permit specificdisease models such as.

In some embodiments, human iPSC can be incorporated into themicrofluidic device, and in certain embodiments, the iPSC can bedifferentiated from a pluripotent state into hepatocytes. iPSC is arenewable cell source that can be easily obtained from academic labs,public cell banks and commercial vendors. iPSC allow forgenotype-specific studies (including individual patients) to beperformed routinely, since the cells are sourced from individual childor adult donors. Further, the iPSC can be modified by established andemerging genetic engineering techniques such that the integration ofbiosensors can be specifically targeted within the cell genome.

In some embodiments, the tissue contained within the microfluidic devicemay exhibit zonation. For example, in liver models, functional gradientor metabolic zonation of the hepatocytes can be established along theacinar sinusoidal units. Different functions (e.g., protein synthesis,ammonia detoxification, drug metabolism) are carried out in the variousmetabolic zones which can lead to unintentional site specific drugtoxicity. For example, acetaminophen exhibits site specific drugtoxicity. Zonation can be measured with existing sensors for oxygen andpH across the long dimension of the device.

Certain embodiments are directed to methods for establishing tissuetypes in a microfluidic device, and such method may include“self-assembly,” which refers to spontaneous formation of tissue-likeorganization. In some embodiments, the method may include of step ofintroducing hepatocytes into the microfluidic device having one or moreinternal compartments having one or more surfaces that are coated withcollagen or another extracellular matrix protein that allows adherenceof the hepatocytes to the microfluidic device. The hepatocytes attach tothe collagen coated surfaces. In some embodiments, the hepatocytes maybe incubated in the microfluidic device for about 12 to about 16 hours.During this time, the hepatocytes may form in vivo-like ‘cords’ or rowsof 2-4 hepatocytes that vary in width and length and cover the surfacesof the device. See FIG. 2. The method may further include introducingendothelial cells, immune cells, and combinations thereof into themicrofluidic device after the hapatocytes have been incubated.Endothelial cells adhere to and spread over the hepatocytes and mayinfiltrate the spaces between the hepatocyte cords. In some embodiments,the microfluidic device may be incubated for about 1 to about 12 hoursafter introducing endothelial cells and/or immune cells into themicrofluidic device. After incubating the endothelial and/or immunecells, the method may include the step of introducing a preparation ofviscous collagen type-1 into the microfluidic device. Such preparationsmay include stellate cells. In particular embodiments, the method mayinclude polymerizing the collagen into a rigid state by inverting thedevice after introducing the collagen preparation to maintain aseparation between the stellate cells and hepatocytes, immune, andendothelial cells. See FIG. 2. After polymerizing has occurred, themethod may include contacting the hepatocytes, immune, and endothelialcells by inverting device to produce self-assembled tissue. See FIG. 2.The microfluidic device may then be attached to a microfluidic pump forthe application of flow medium over the self-assembled tissue.

In some embodiments, the method may include flowing flow medium throughthe microfluidic device with self-assembled tissue for about 1 to about14 days. During this time the cell types migrate and self-assemble intodistinct layers forming liver like tissue. The layers may includehepatocytes attached to the surfaces of the device in cord formations.Stellate cells may migrate to the hepatocyte layer, and endothelialcells may leave the hepatocyte layer to form a loose interconnectedlayer above the hepatocytes. This arrangement has similarities to theorganization and architecture of the liver sinusoidal unit, afundamental component of liver organization and function.

The microfluidic devices of various embodiments may have a similararchitecture to that described above. For example, in certainembodiments, the microfluidic devices described above may includehepatocytes attached to the surfaces of the device in cord formations,stellate cells associated with the hepatocyte layer, and endothelialcells in a loose interconnected layer above the hepatocytes as depictedin FIG. 2. The four human cell types discussed above contribute to liverhealth, and therefore toxicity response and disease phenotype. Inparticular embodiments, human hepatocytes (primary or iPSC-derived),human endothelial cells (EAhy.926 or similar or primary), human immunecells (monocytes U937 or similar or primary), and human stellate cells(LX-2 or similar or primary) may be used in particular embodiments ofthe invention, although other cell types may be incorporated into themicrofluidic devices to reproduce other tissue types. In suchembodiments, the primary hepatocytes may retain metabolic capacity notfound in immortalized hepatic cell lines. Human stellate cells reproducefibrotic liver scarring, and immune cells reproduce immune-mediatedtissue damage both important mechanisms of toxicity.

FIG. 2 shows the organization of cells as discussed above. Panel a showsa cross-section diagram based on confocal images of the typical cellorganization on day 1 after cell seeding of the hepatocytes, Kupfferimmune cells (U937), endothelial (EAhy.926) and Stellates (LX-2).Hepatocytes are first seeded on a layer of collagen. Endothelial cellsand U937 are seeded 18-24 hours after hepatocytes and initially localizebetween the hepatocytes. LX-2 Stellates are seeded and are initially,evenly dispersed in the collagen layer. Panel B shows a diagram oftypical cell organization 7 days after cell seeding, when endothelialcells and Stellate cells are localized both between hepatocytes and in alayer above the hepatocytes. The schematics in panels A and B of FIG. 2were abstracted from the scans of 26 image planes 10 μm apart startingat the hepatocyte layer.

FIG. 3 show confocal images of a microfluidic device in which a subsetof the total seeded cells (˜20%) are sentinel cells stably expressing afluorescence-based biosensor and the remaining cells are unmodified. Inpanel A, primary hepatocytes self-organized into cords by day 7 areidentified by inhibiting cannicular transport of a dye (CMFDA, green).The other cell types self-assemble relative to the hepatocytes.Mitochondria are labelled with TMRE (red) and cell nuclei with hoechst(blue). In panel B, labeled hepatocyte chords (CMFDA, green). The cellsare under constant flow at ca. 5 μl/hr with the influx port at the topand the efflux at the bottom. The red box represents the size of a 20Xfield shown in panel A within the chamber and the dashed yellow boxshows the region from which multiple image fields were collected.Approximately 200,000 total cells are present in the entire chamber,added in physiological ratios of hepatocytes and non-parenchymal cells.

The microfluidic devices of various embodiments may include a flow pumpassociated with an inlet to the housing and an outlet that allow flowmedia to leave the microfluidic device. In some embodiments, the pumpmay draw flow medium from a primary reservoir and pump the flow mediathrough the inlet into the housing where the flow medium can contact thecells and tissues in the microfluidic device. The flow media may exitthe microfluidic device through an outlet port. The outlet port may beassociated with a second reservoir where flow medium is collected orsample reservoirs, where samples are collected. In some embodiments, theflow medium may be collected in the primary reservoir and pumped backinto the microfluidic device. In such embodiments, the flow medium maybe mixed with nutrients and other materials necessary to maintain thecells and tissues in the microfluidic device before being pumped backthrough the device. Flow of flow media is important for maintainingcells in 3D tissue and tissue models, to induce in vivo-likeorganization and function, including oxygen zonation, and to deliverreagents to the cells. In some embodiments, the microfluidic devices mayinclude one or more ports to load cells and introduce nutrients, drugs,test substances, and other materials into the microfluidic devicewithout modifying the flow of flow media through the device.

The loss of hepatocyte functionality occurs within days in the absenceof additional cell types and a biological matrix such as collagen. Theunique combination of cell types, extracellular matrix proteins and flowthrough culture conditions in the invention has demonstrated retentionof hepatocyte cell polarity and metabolism for up to 28 days (Seefigures in associated document). The ability of the SALA model tomaintain function long-term allows for detection of chronic drugtoxicities and disease phenotypes that manifest over weeks as opposed toacute toxicities that generally occur within days.

The microfluidic devices of various embodiments describe above allow fordirect 3D imaging of fluorescent protein-based biosensors and otherfluorescent probes, as well as brightfield imaging of cell morphology.This innovation allows for non-destructive monitoring 28 days or more tomeasure specific molecular events within cells. The molecular eventscorrespond to clinically significant signatures of drug toxicity anddisease state. The molecular mechanisms of toxicity (“MOT”) currentlymonitored in cells and are associated with adverse drug reactionsinclude oxidative stress, mitochondrial dysfunction, bile canalicularefflux inhibition, immune mediated toxicity, and fibrotic scarring. Manyother MOTs can be evaluated through the use of fluorescent biosensors.In addition, the in vitro liver model can also be used to model diseasestates, and disease associated molecular mechanisms of action (“MOA”),for the testing of potentially therapeutic drugs.

Certain embodiments are directed to systems including the microfluidicdevices described above and a computer in connection with the one ormore sensors, imaging devices, or combinations thereof. The computer maybe configured to receive data from the optical system and to store atleast a portion of the received data in one or more memory components asat least one database. The computer may be further configured to analyzethe images and sensor data received by the memory components andidentify particular components of the images such as, damaged, cells,biomarkers, metabolites, and the like and combinations thereof.

Embodiments of the invention include methods for testing the response ofcells and tissues enclosed in the housing of the microfluidic devices toexposure to a test substance. Such methods may include the step ofintroducing one or more reference substances into a microfluidic devicewith established tissue architecture and detecting a response to the oneor more reference substances. Detecting can be carried out by variousmeans including for example, capturing images of the tissue afterexposure, detecting biomarkers associated with toxicity or diseasesstates, and the like and combinations thereof. In some embodiments, themethods may further include constructing a computational model topredict human liver response to exposure to the reference substance.Such modeling may include generating pharmacokinetic and pharmacodynamic(PK/PD) data, identifying toxic substance, and determining efficacy ofreference substances for improving a diseased state.

In some embodiments, such methods may include providing a microfluidicdevice having established architecture of the liver sinusoidal unitssuch as those described above, introducing one or more test materialsinto microfluidic device, and detecting one or more analytes in theeffluent of the microfluidic device. Other methods of embodimentsinclude providing a microfluidic device having established architectureof the liver sinusoidal units such as those described above, introducingone or more test materials into microfluidic device, and capturingimages of the architecture of the liver sinusoidal units afterintroducing the test material into the microfluidic device, and in someembodiments, such images may include analytes. In still otherembodiments such methods may include both detecting an analyte andcapturing images. The analytes of various embodiments may vary and canbe, for example, metabolites of the test material, biomarkers,fluorescent markers from test materials, biomarkers and metabolitesassociated with a diseased state, cellular debris, and the like andcombinations thereof.

In some embodiments, the method may incorporate the use of multi-colorfluorescent protein-based biosensors to track physiological functionsand their perturbation in real-time. The biosensor cells, also referredto as “sentinel cells,” can be created, for example, by integration ofthe genetic sequence of the biosensor into the host cell genome vialentiviral transduction. Such biosensors are expressed in cells throughnormal transcription/translation. In various embodiments, the biosensorscan be used to monitor cell movement, cell division, apoptosis, reactiveoxygen, and calcium flux in mitochondria. Biosensors are stable over thelifetime of cells whereas fluorescent probe dyes can become chemicallymodified to a non-fluorescent state, and can diffuse, or even be pumpedfrom the cells within hours to days. Protein biosensors have less effecton cell health compared to dyes that are known to produce cytotoxicoxygen radicals when illuminated (photobleaching). The combination oflentiviral delivery, availability of multiple colors of fluorescentproteins and the user-controlled addition of cells to the model lendsthe flexibility needed to monitor distinct events in different celltypes. For example, the generation of reactive oxygen in hepatocytes canbe observed while monitoring initial steps leading to fibrotic scarring,such as the stimulation of stellate cell division. Additionally, thebiosensors are sensitive and reversible such that abnormal levels ofcalcium (or other ion) fluxes within a cell are detected, but normalcalcium (or other ion) distributions will be indicated when restored tonormal levels in the cell.

In some embodiments, the data collected from the microfluidic devicescan include biochemical, mass spectroscopy, biosensor cells, readoutsfrom other fluorescent probes, and morphological readouts.Interpretation of the results as indications of potential humanliabilities, or therapeutic benefits may include computational modelsthat can predict human drug interactions from the data provided by theorgan model. To construct these predictive models data from preclinical,clinical, and post-market drug trials can be compared with the readoutsof the microphysiology organ model in a microphysiology database. Thedrug trial data can be collected from public databases and literaturereferences. These combined data can be used to develop classificationmodels such as random forests, support vector machine, and others thatwill predict human liver-drug interactions from the microphysiologyliver-drug interactions measured from the microfluidic devices.

From the foregoing, it will be appreciated that various embodiments ofthe present disclosure have been described for purposes of illustration,and that various modifications may be made without departing from thescope and spirit of the present disclosure. Accordingly, the variousembodiments disclosed are not intended to be limiting, with the truescope and spirit being indicated by the following claims. The presentdisclosure is not to be limited in terms of the particular embodimentsdescribed in this application, which are intended as illustrations ofvarious aspects. Many modifications and variations can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. Functionally equivalent methods and apparatuseswithin the scope of the disclosure, in addition to those enumerated inthis disclosure, will be apparent to those skilled in the art from theforegoing descriptions. Such modifications and variations are intendedto fall within the scope of the appended claims. The present disclosureis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which such claims are entitled. It isto be understood that this disclosure is not limited to particularmethods, reagents, compounds, or compositions, which can, of course,vary. It is also to be understood that the terminology used in thisdisclosure is for the purpose of describing particular embodiments only,and is not intended to be limiting.

EXAMPLES

Efflux media was collected for measurements of organ model health usingassays for lactate dehydrogenase (LDH) release, and albumin, and ureasynthesis. LDH is released from cells under conditions of cytotoxicdamage. Albumin and urea are secreted from functional hepatocytes. Themedia efflux was also used to monitor the presence of drug and metabolicproducts for PK. When compounds are included in the microfluidic media,hepatocytes are exposed to the compound and often biotransform them to achemically distinct product. Mass spectrometry is a specific andsensitive method to observe these changes. Typically, ca. 15 microlitersof media is collected for each biochemical and mass spec measurement(flow rate=5 microliters per hour.)

Example 1

Long-term health and stability of hepatocytes was tested using amicrofluidic device with self-assembly liver acinus (“SALA model”), andthe results are provided in FIG. 4. Lactate dehydrogenase (LDH) leakagemeasurements in efflux media are stable from day 2 to day 28 isillustrated in panel A. Panel B shows that urea (top line) and albumin(lower line) output in the efflux media are relatively stable andphysiological for over 28 days. Results presented as mean ±SD from 3(days 3, 10 and 17) or 4 devices (days 7, 13 and 25).

Example 2

Liquid chromotorgraphy—mass spectroscopy (“LC-MS”) of effluent wascarried out and a chromatogram showing metabolic clearance andmetabolite production of SALA model in the microfluidic device on day 10is provided in FIG. 5. Panel A shows the result of introducing 5 μMtestosterone in influx media. Panel B shows testosterone in efflux mediacollected over 24 hr at 5 μl/hr flow and demonstrates 27% oftestosterone exits unchanged through the device as calculated as a ratioof the mass area (MA) under the curves. This value is concordant withtestosterone clearance. Panel C shows Cyp3A4 signature metabolite6-β-hydroxytestosterone in efflux media collected at 5 μl/hr for 24 hr.

Example 3

Live cell monitoring of hepatocellular cytochrome C release biosensorand biochemical assays performed on the efflux media demonstrates timeand dose dependent toxicity is shown in FIG. 6 using the SALA model in amicrofluidic device. Troglitazone (28 or 180 μM), or caffeine (600 μM,negative control) was introduced into the microfluidic device for 0-28days. Troglitazone induces apoptosis in the hepatocytes as indicated bythe release of the cytochrome c biosensor in the sentinel cells with aT½ of ˜14 days (28 μM) or ˜1 day (180 μM) as illustrated in panel A,while caffeine shows no significant effect over 28 days as illustratedin panel B. These data show that troglitazone induces a decline inalbumin secretion with a T½ of ˜11 days (28 μM) or ˜1 day (180 μM),while caffeine shows no significant effect. Troglitazone induces adecline in urea secretion with a T½ of ˜12 days (28 μM) or ˜3 days (180μM) as illustrated in panel C, while caffeine shows no significanteffect as illustrated in panel D. Troglitazone induces release of LDHwith a peak loss at ˜13 days (28 μM) or ˜4 days (180 μM), while caffeineshows no significant effect. All values are means from two devices.

Example 4

Acute nimesulide toxicity was assessed using the SALA model in amicrofluidic device and the results are provided in FIG. 7. Nimesulide(210 μM) induces apoptosis, as indicated by cytochrome C biosensorrelease, with a T½ of ˜3.5 days as illustrated in panel A. LDH leakagealso occurred along with induction of apoptosis, and decreases inalbumin and urea synthesis, with peak at ˜5 days and T½ of 3 and 4 days,respectively, were also observed as illustrated in panel B. All valuesrepresent the mean of two liver devices.

Example 5

Active immune function was observed using the SALA model in amicrofluidic device, and the results are provided in FIG. 8.Lipopolysaccharide (LPS) activated U937 cells release hepatotoxicTNF-alpha with an EC50 of 0.7 μg/ml as illustrated in panel A. IncreasedLDH leakage was evident in SALA models treated 3 and 5 days with 200 μMtrovafloxacin plus 1 ug/ml LPS or 7 and 10 days with 200 μMtrovafloxacin as illustrated in panel B. The shift in toxicity isevident as a decrease in hepatocyte area measured in real-time on days 5and 7 in the SALA models exposed to 200 μM trovafloxacin plus 1 ug/mlLPS or 200 μM trovafloxacin, respectively, as illustrated in panel C. Nohepatocyte area decrease is evident in 600 μM levofloxacin plus 1 ug/mlLPS or 600 μM levofloxacin treated devices as expected. The decrease inhepatocyte area with trovofloxacin plus LPS treatment can be correlatedto an increase in propidium iodide uptake. The results are the mean of 2devices per treatment group.

Example 6

LX-2 stellate cell activation and migration in SALA was tested using theSALA model in a microfluidic device, and the results are provided inFIG. 9. In response to 30 nM methotrexate treatment over 21 days, LX-2stellate cells divide and transmigrate from the upper matrix in the SALAmodel and accumulate in the lower hepatocyte layer at an acceleratedrate compared with vehicle (1.0% DMSO) treatment measured by imageanalysis of the total SALA model volume as illustrated in panel A.Expression of human α-SMA and collagen type 1A2 proteins in LX-2stellate cells on day 21, quantitated by image analysis is increasedwith 30 nM methotrexate treatment, relative to vehicle control isillustrated in panel B.

Example 7

Bile efflux in vehicle and troglitazone treatment was tested using theSALA model in a microfluidic device, and the results are provided inFIG. 10. Panel A shows CMFDA fluorescence monitoring in hepatocytes at10 minute intervals for 60 minutes in a following 10 day treatment withvehicle control (1% DMSO) or troglitazone (50 μN). Normal uptake andefflux of the dye is evident in vehicle compared to the slower uptakeand inhibition of efflux in the troglitazone treated device. Panel Bshows a 20X image of vehicle control treated liver model demonstratingnormal efflux (white canaliculi) at 60 minutes. Panel C shows the BSEPinhibitor troglitazone (50 μM) d increased hepatocyte retention of dyeat 60 minutes. Bile efflux is demonstrated on Day 21 in the SALA underuntreated media flow as illustrated in panel D.

Correlation of propidium iodide (PI) uptake with cell area ofhepatocytes is illustrated in FIG. 11. The cytochrome C biosensor isused to measure hepatocyte area in order to perform real-timemeasurements of apoptosis. Trovafloxacin (200 μM) co-treated with 1ug/ml LPS induces toxicity in hepatocytes as noted by a reduction incell area and increased PI uptake when compared to vehicle (1% DMSO).

Example 8

LX-2 stellate cell activation was observed using the SALA model in amicrofluidic device, and the results are provided in FIG. 12.Immunofluorescence image of a SALA liver exposed to vehicle for 21 days,fixed and reacted to α-SMA antibody is provided in panel A. Minimalα-SMA expression (false colored red) is evident in green stellate cells(white arrow) or in hepatocytes (yellow arrow). Panel B showsimmunofluorescence imaging of a SALA liver treated 21 days with 30 nMmethotrexate, fixed and reacted against α-SMA antibody. An increased inα-SMA expression (false colored red) is evident by the increasedyellow/white color in stellate cells (white arrow) and red color inhepatocytes (yellow arrow). Day 21 Immunofluorescence images of a SALAliver vehicle treated 21 days, fixed, and reacted against α-HumanCollagen type 1/2 is provided in panel C. Expression of collagen isidentified as increased yellow (white arrow) in stellate cells which arefalse colored red. Not all stellate cells express high levels ofcollagen (orange arrow). Nuclei are false colored blue in all images.

Example 9

Demonstration of the uniformity of flow using the SALA model in amicrofluidic device is shown in FIG. 13. On Day 7 post-cell seeding,injection of an ˜20 μL bolus of 0.1 mg/mL Alexa Fluor® 647-dextran (10KDa) as a tracer was followed by washout with media flow at 5 uL/hr. Thedevice was imaged at 1 hour intervals on the InCell6000 at 10×magnification. Panel A shows an image sequence (only a subset is shown)showing the uniformity of the influx and washout of fluorescent dextranacross the width of the microfluidic device. Panel B shows a graph ofthe average dextran intensity in the interrogation region (dashed yellowbox, A. 6 hrs.) normalized to the maximum value.

Example 10

Statistical validation of biosensors in hepatocyte sentinel cells wascarried out and the results are provided in TABLE 1. A panel ofbiosensors was delivered through lentiviral transduction in multiplefluorescent protein colors initially validated in microplates for anacute response. An SSMD level of ≧2 is considered a statistically validresponse.

TABLE 1 Color Control Conc Biosensor Options Compound (μM) SSMDNuclear/cell position Blue Cell Tracking and — — (Histone H2B) GreenProliferation Red (Kupffer and Stellate) Cytochrome C Green Menadione100 >2 (at 5 hr)   Release: Apoptosis Red Reactive Oxygen GreenMenadione 100 >2 (at 2.5 hr) Species in Mito. Mitochondrial GreenMenadione 50 >2 (at 4.5 hr) Calcium Uptake Steatosis (Label-Free) WhiteTamoxifen 75 >6 Bile canalicular efflux Green Troglitazone 50 11 (CMFDA)Oxidative Stress in Blue Nefazadone, 75 In develop Mito. & CytoplasmGreen Menadione 50 Red

Example 11

MDA-MB-231 cells exhibit two distinct subpopulations in the SALA modelin a microfluidic device. One population that is invasive andproliferates and another that is quiescent. These results are providedin FIG. 14 Pane A shows the device a day 3 (10X) with a population ofred quiescent MDA-MB-231 cells residing in tissue layer of naive (gray)and sentinel (green) hepatocytes. Panel B shows the device at day 9(10X) with a population of red quiescent MDA-MB-231 cells residing intissue layer of naive (gray) and sentinel (green) hepatocytes. Panel Cshows the device at day 9 (10X) at the same location as shown in panel Bbut focused on invasive MDA-MB-231 cells in collagen layer abovehepatocytes. Green label hepatocytes not shown. Panel D shows red labelMDA MB 231 cells collected in the hepatocyte layer from Day 0-16 andcounted in the efflux media from Day 8-Day 20. A slow increase inproliferation is evident in the cells residing in the hepatocytes but arapid increase is evident in the collected efflux. This demonstrates theuse of the SALA liver model as a metastatic breast cancer niche tofollow the activity of labeled MDA-MB-231 cells, as well as other cancercells, and the response of the liver micro-environment.

Example 12

Nefazodone induces oxidative stress in the SALA model in a microfluidicdevice, and the results are provided in FIG. 15. Panel A is a montage ofbrightfield images demonstrating flexibility of HCA instruments tocollect and analyze any device layout. Panel B shows biosensortransduced hepatocytes that are viable and well distributed in device.Panel C shows nefazodone induces oxidative stress in the hepatocytes,which can be visualized (½ images presented) as a decrease inmitochondrial function (Red) and increase of ROS generation (Green) at 0to 15 hr treatment period. Image processing at each time point provideskinetic physiological responses is provided in Panel D.

Example 13

The performance of 4 selected biosensors was tested. One fluorescentreadout for bile efflux and control compounds correctly identifiedhepatotoxic compounds and the mode of toxicity. The results are providedin TABLE 2:

TABLE 2 Drug Cmax Human/Animal Metabolism MOT Our Toxicology dependenceResults Negative Weak or late positive Strong Positive Trazodone 5 uMextremely rare liver High No known ROS (in use) injury clearance drugliver tox Calcium Mild ↑transaminases, uptake self eliminating Mito.0.21 membrane incidents/million potent. prescriptions Apoptosis bileefflux Nefazodone 0.9 uM Acute hepatitis High Mitochondrial ROS(withdrawn Centrilobular (zone 3) clearance Inhibitor Calcium US)necrosis drug, toxic ROS uptake Cholestasis, apoptosis intermediatesGeneration Mito. 105 incidents/million Bile Efflux membraneprescriptions Inhibition potent. Apoptosis bile efflux Troglitazone 1.8uM Moderate to severe Impaired Mitochondrial ROS (Withdrawn) ↑ALT, ASTclearance dysfunction Calcium Variable necrosis, BSEP inhibitor uptakehepatocellular (bile efflux Mito. damage, inhibition) membranecholestasis, potent. inflammatory Apoptosis response bile efflux Up to1000 incidents/million prescriptions Menadione Rat Infants: menadione Notoxic ROS ROS (positive 100 mg/kg injections produce intermediatesMitochondrial Calcium control) liver toxicity with known Inhibitionuptake hyperbilirubinemia Calcium Mito. Rat Toxicity (Kidney, uptakemembrane Heart, Liver, Lung) potent. IV infusion: Liver: Apoptosisinflammation, bile efflux degeneration, vacuolization and necrosis MOTidentified ROS in liver, calcium uptake into hepatocytes massive livernecrosis in GSH depleted rats

What is claimed is:
 1. A microfluidic device comprising: a housinghaving at least one inlet and at least one outlet; extracellular matrixproteins disposed on at least one surface of the housing; cells of atleast two types associated with the extracellular matrix proteinsforming three-dimensional structures within the housing; and a flowmedium contacting the cells.
 2. The device of claim 1, wherein the cellsof at least two types are selected from the group consisting of primaryliver cells, cultured liver cells, induced pluripotent stem cells,primary hepatocytes, endothelial cells, immune cells, stellate cells,and combinations thereof.
 3. The device of claim 1, wherein the cells ofat least to types are human hepatocytes, human endothelial cells, humanimmune cells, and human stellate cells.
 4. The device of claim 1,further comprising genetically modified cells expressing a geneticallyencoded fluorescence based biosensors.
 5. The device of claim 4, whereinexpression of the genetically encoded fluorescence based biosensor isdependent upon calcium levels, pH, glutathione levels, mitochondrialcalcium levels, oxidative stress, or reactive oxygen species.
 6. Thedevice of claim 4, wherein expression of the genetically encodedfluorescence based biosensor is in response to apoptosis, change inmitochondrial membrane potential, cell proliferation, free calcium ionconcentration, cell motility, and oxidative stress response.
 7. Thedevice of claim 1, wherein the one or more three-dimensional structureshave established zonation.
 8. The device of claim 1, wherein thethree-dimensional structures are liver acini.
 9. The device of claim 1,further comprising a pump fluidly connected to the inlet configured topropel flow medium through the housing.
 10. The device of claim 1,wherein the housing comprises one or more chambers and one or morepassages fluidly connecting the chambers.
 11. The device of claim 10,wherein the extracellular matrix proteins are disposed in the chambersof the housing.
 12. The device of claim 1, further comprising a sensorpositioned to detect analytes in effluent exiting the housing.
 13. Thedevice of claim 1, further comprising an imager positioned to image thethree-dimensional structures.
 14. The device of claim 1, furthercomprising one or more ports positioned to introduce substances into theflow medium.
 15. A method for self-assembly of liver acini comprising:providing a surface coated with an extracellular matrix protein;contacting the surface with hepatocytes; incubating the surface forabout 12 to about 16 hours; contacting the surface with endothelialcells, immune cells, or combinations thereof; incubating the surface forabout 1 to about 12 hours; and contacting the surface with polymerizedcollagen.
 16. The method of claim 15, wherein the surface is disposedwithin a microfluidic device.
 17. The method of claim 16, whereincontacting the surface with polymerized collagen comprises introducingcollagen into the microfluidic device; inverting the microfluidicdevice; polymerizing the collagen; and inverting the microfluidicdevice.
 18. The method of claim 15, wherein the hepatocytes furthercomprise genetically modified hepatocytes expressing a geneticallyencoded fluorescence based biosensors.